Journal of General Microbiology (1988), 134, 22 13-2229.
Printed in Great Britain
2213
Structure and Molecular Species Composition of Three Homologous Series
of a-Mycolic Acids from Mycobacterium spp.
By K E N J I K A N E D A , ' * t S A D A O I M A I Z U M I , ' S E I K O M I Z U N O , '
T S U N E K O BABA,3 M I C H I O T S U K A M U R A 4 A N D I K U Y A Y A N 0 3
Department of Bacteriology, Niigata University School of Medicine, Asahimachidori I ,
Niigata, Japan
'Soai WomenS College, Osaka, Japan
Department of Bacteriology, School of Medicine, Osaka City University, Asahimachi 1-4-54,
Abeno-ku, Osaka, Japan
4The National Chubu Hospital, Obu, Aichi, Japan
(Received 8 December 1987; revised 15 February 1988)
Three homologous series of a-mycolic acids (dicyclopropanoyl acids, monocyclopropanoyl
monoenoic acids and dienoic acids) from 16 rapidly growing and 14 slowly growing
mycobacteria were separated by argentation thin-layer chromatography and analysed by gas
chromatography/mass spectrometry of their trimethylsilyl ether derivatives. Mycobacterial
species were separated into five groups. Strains of group A contained similar amounts of even
and odd carbon-numbered dienoic acids, with a methyl branch on the odd acids and a C24-aunit, as typified by Mycobacterium fortuitum and M . chitae. Group B strains possessed similar
amounts of even carbon-numbered dicyclopropanoyl a-mycolic acids and odd carbon-numbered
unsaturated acids with Cz2-and Cz4-a-units,as found in M . phlei and M . diernhoferi. Group C
strains contained mainly even carbon-numbered dicyclopropanoyl acids with Ct2- and
C2,-a-units, as shown by M.vaccae and M . aurum. Group D strains possessed mainly odd
carbon-numbered dienoic acids with a methyl branch and a C,,-a-unit, as seen in M . triviale and
M . nonchromogenicum. Group E strains had mainly even carbon-numbered dicyclopropanoyl
acids with C24-or C2,-a-units, as found in M . avium and M . tuberculosis. Many rapidly growing
mycobacteria also produced a'-mycolic acids which were shorter in the length of the main
carbon chain but whose a-units were the same as those in a-mycolic acids from the same species.
These a'-mycolic acids had either one or two double bonds and showed variations in both their
unsaturation and overall size, which may be useful in taxonomic studies.
INTRODUCTION
The analysis of subclasses of mycolic acids, the most characteristic cell wall component of the
acid-fast bacteria, by thin-layer chromatography (TLC) (Daffk et al., 1983) or by twodimensional TLC (Minnikin, 1982; Minnikin et al., 1980, 1984, 1985) has shown that several
characteristic patterns are useful for the classification of mycobacteria. As for the structural
determination of mycobacterial a-mycolic acids, several species such as Mycobacterium phlei
(Kusamuran et al., 1972), M . smegmatis (EtCmadi et al., 1967), M . chelonae (Minnikin et al.,
1982), M . fortuitum (Lacave et al., 1987), M . tuberculosis (Minnikin & Polgar, 1967),
Present address: Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University,
Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan.
Abbreviations: AgN0,-TLC, argentation thin-layer chromatography; TMS, trimethylsilyl; monon,
monocyclopropane ; d i n , dicyclopropane.
0001-4564 0 1988 SGM
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K . K A N E D A A N D OTHERS
M .parafortuitum (Lankelle & Lankelle, 1970), M. avium (Lamonica & Etkmadi, 1967), M. kansasii
(Etemadi, 1967) and M. marianum (scrofulaceum)(Bruneteau & Michel, 1968) (for a review, see
Minnikin, 1982) have been examined by mass spectrometry (MS) of their degradative products
after pyrolysis or ozonolysis and by nuclear magnetic resonance spectroscopy. By isolating each
molecular species of mycolic acid using high-performance liquid chromatography, a-mycolic
acids of M.smegmatis (Danielson & Gray, 1982) and M. tuberculosis (Qureshi et al., 1978) were
successfully analysed for each molecular species using MS. We have clarified the structure and
the molecular species composition of a-mycolic acids in Nocardia spp., Rhodococcus spp. and
Mycobacterium spp., using gas chromatography (GC), GC/MS and mass chromatography. The
molecular species composition is characteristic for each species, and GC/MS and mass
chromatographic analysis of mycolic acids is very precise and informative (Yano et al., 1978;
Toriyama et al., 1978; Kaneda et al., 1986a,b). Three different homologous series of
mycobacterial a-mycolic acids have been reported, dicyclopropanoyl acids,
monocyclopropanoylmonoenoic acids and dienoic acids (Minnikin, 1982;Daffk et a!., 1983). In
a previous paper on the molecular species composition of 25 mycobacterial species (Kaneda et
al., 1986b), we did not distinguish cyclopropane rings from double bonds in the main chain
portion of a-mycolic acids, because the mass spectra of a-mycolic acids did not permit this
information. In this study, we have differentiated these three homologous series of a-mycolic
acids in 30 mycobacterial species. Their distribution and molecular species characteristics have
been studied using argentation thin-layer chromatography (AgN0,-TLC), GC/MS combined
with a hydrogenation technique and mass chromatography.
METHODS
Strains and culture conditions. The following 16 rapidly growing species were studied. Photochromogenic
species: M . uaccae VA-1 and M . parafortuitum PA-1, PA-4,19686. Scotochromogenic species: M . phlei 14002; M .
rhodesiae RHO-I ; M . gilvum GI-I ; M . duvalii DU-2, 29505; M . aurum AU-1; and M . thermoresistibile 01028,
laboratory strain. Nonchromogenic species: M . chelonae R-1, R-2, 2201 1 (subsp. abscessus), 19009 (subsp.
chelonae);M . smegmatis Takeo, Rabinobitz; M . chitae CH-2, CH-3; M .fortuitum F-6, 18001, 18112, E-11592 and
E-11620 (subsp. acetamidolyticum); ' M .peregrinum' PE-4; M . porcinum 19506; M . diernhoferi 41002, 41004; M .
pulveris 33505 ; and M . agri 90002,90012. Cultures of the above species were incubated on a shaker at 30 "C until
mid-exponential phase (about 3-5 d) in medium (pH 7.0) containing 1% (w/v) glucose, 0.5% peptone and 0.2%
yeast extract, except that M .fortuitum subsp. acetamidolyticum was cultured for 7 d and M . thermoresistibile was
grown at 37 "C for 7 d.
The following 14 slow growing species were studied. Photochromogenic species : M . kansasii laboratory strain;
M . marinum 08010; and M . simiae 93001. Scotochromogenic species : M . gordonae T-12109. Nonchromogenic
species : M . terrae 38016;M . nonchromogenicum09003; ' M . novum' 240 18; M . triviale 370 14; M . avium 11016; M.
intracellulare 13023; M . ulcerans 28504; M . shimoidei43501; M . bouis BCG, Ravenel; and M . tuberculosis H3,Rv.
These cultures were grown for 3-5 weeks at 37 "C in Sauton medium, pH 7.2 or on slants containing Ogawa egg
medium, which contained 100 ml of a basal solution (1 %, w/v, sodium glutamate and 1%, w/v, KH2P04),200 ml
whole egg, 6 ml glycerol and 6 ml 2% (w/v) malachite green solution.
Preparation of fatty acid methyl esters. Whole cells, harvested by centrifugation, were hydrolysed with 15% (w/v)
KOH at 90 "C for 3-4 h. After acidification with HCl, the fatty acids liberated were extracted with n-hexane and
subsequently methylated in 5-10 ml benzene/methanol/concentratedH2S04 (10 :20 :1, by vol.) at 90 "C for 1.5 h.
TLC and AgN03-TLC.The fatty acid methyl esters were developed on a TLC plate of Silica gel G (Analtech)
with n-hexane/diethyl ether (4 :1, v/v). After locating with iodine vapour each mycolic acid subclass was recovered
with chloroform. Isolated a-mycolic acid methyl esters were further separated on a 10% (w/v) AgN0,-TLC plate
of silica gel 60G (Merck) with n-hexane/diethyl ether (94 :6, v/v); this developing procedure was repeated four
times. The spots were visualized with charring after being sprayed with 50% (v/v) H2S04. For recovery, TLC
samples were located with iodine vapour and AgN0,-TLC samples were located with 2',7'-dichlorofluorescein.
Hydrogenation experiments. a-, a'- or a a'-mycolic acids separated on TLC, or one class of a-mycolic acids
separated on AgN0,-TLC, were recovered with chloroform and hydrogenated to distinguish double bonds from
cyclopropane rings in the molecules;double bonds can be hydrogenated in neutral solvents, whereas cyclopropane
rings can be hydrogenated only in acidic solvent systems. The sample was hydrogenated in chloroform/methanol
(2 :1, v/v) with a few milligrams of platinum oxide (PtO, : Adams catalyst) for 1 h at room temperature or in glacial
acetic acid for 4 h at 40 "C to cleave the cyclopropane rings (Kaneshiro & Marr, 1961). After removal of the
catalyst by filtration, the solvent was evaporated off.
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Mycobacterial mycolic acids
2215
Preparation of trimethylsilyl (TMS)-ether derivatives of mycolic acid methyl esters. For GC and GC/MS analysis,
a- and a'-mycolic acid methyl esters before or after the hydrogenation were converted to the TMS derivatives,
which are stable at high temperatures (Yano et al., 1978; Toriyama et al., 1978). Pyridine (0.1 ml) and
bistrimethylsilyl-trifluoroacetamide(0-2 ml) were added to the dried methyl mycolate (1-5 mg) and incubated at
70 "C for 20 min. The solvent and by-products were evaporated off.
GC,G C / M S and m a s s chromatographic analysis of the TMS-ether derivatives of mycolic acid methyl esters. The
procedure has been described in detail previously (Kaneda et al., 19866). TMS derivatives of methyl mycolate
were analysed by GC (Hitachi 063) with a glass column (0.4m x 3mm, 1% OV-101 on Gaschrom Q, column
temperature 330 "C isothermally) and subsequently by a GC/MS system (Hitachi M-80B) with the same column
(the ionization voltage was 20 eV and the accelerating voltage, 3 kV). The mass spectra were represented as the
average value of several scans around the peak top on the chromatograms of the total ion current. Mass
chromatograms were obtained by monitoring [M - 15]+ions of the TMS derivatives of each carbon-number class
of mycolic acid methyl esters. On mass chromatograms each molecular species could be shown as an independent
peak, and by measuring its peak area the molecular species composition and the average carbon number
[ =E(carbon number x percentage/l00)] could be determined. As well as determining the overall size of mycolic
acids, the size of the socalled CZ2-,CZ4-and C2,-a-units was determined by mass chromatogram monitoring of
ions resulting from cleavage between the second and third carbon in the a-mycolic acid derivatives.
RESULTS
Subclass composition of mycolic acids
On TLC plates spots corresponding to non-polar fatty acid methyl esters were observed close
to the solvent front and below them several spots were seen corresponding to each subclass of
mycolic acid methyl ester. a-Mycolic acids, the most hydrophobic among all the mycolic acid
subclasses,were usually a major component in every mycobacterial species except for M. duvafii
and M . shimoidei, which contained only a trace amount. a'-Mycolic acids, whose main carbon
chains are shorter than those of a-mycolic acids, were present in many rapidly growing species
(all except M . diernhoferi, M. gilvum, M . phlei and M . rhodesiae) and in two slow growers, M.
simiae and M . shimoidei.
Analysis of a-mycolic acids
AgN03-TLC.On AgN03-TLC, purified a-mycolic acid methyl esters both from rapidly and
from slowly growing mycobacteria were further separated (Fig. 1). Each consisted of one or two
components (upper and lower spots). In the rapid growers M. chitae, M .fortuitum, M . smegmatis
and M . chelonae, a lower spot predominated but, except for M. chitae, they also had a small
amount of an upper spot. M . rhodesiae, M. diernhoferi, M . phlei and M . parafortuitum contained
both upper and lower spots, and M. pulveris, M. thermoresistibile, M . aurum, M . vaccae, M .
gifvum and M . agri possessed primarily an upper spot. The slow growers M. nonchromogenicum,
M . terrae, M . triviale and 'M. novum' possessed mainly a lower spot with a very minor upper
component. With the exception of M. bovis BCG, which also contained a smaller amount of a
lower spot, M. tuberculosis, M. kansasii, M . simiae, M . marinum, M . gordonae, M . avium, M .
intracellulare and M . ulcerans contained only an upper spot.
GC. The gas chromatograms of a-mycolic acid derivatives gave generally well-separated
peaks ranging from C70to CB2in rapid growers (Fig. 2) and from C72to Cg6in slow growers
(Kaneda et al., 1986b). The carbon number of the a-mycolic acids in each peak was determined
from its mass spectrum as described later. The a-mycolates from M. parafortuitum, M. rhodesiae,
M . phlei and M . diernhoferi, however, showed gas chromatographic profiles with poorly
separated peaks, probably due to a higher content of odd carbon-numbered acids.
GC/MS. The structure of the molecular species of a-mycolic acids separated by G C was
determined from the mass spectra. From the mass number of the characteristic fragment ions of
the TMS methyl a-mycolate, i.e. [MI+(molecular ion), [M - 15]+(loss of -CH3), [M - 90]+ (loss
of trimethylsilanol), [A]+ (TMS-alkoxy ion resulting from C2-C3 cleavage), [A - 90]+ (loss of
trimethylsilanol from [A]+), [B]+ (TMS-oxymethylene carboxylic acid ester resulting from
C3-C4 cleavage) and [B - 29]+ (loss of CHO from [B]+), the numbers of carbons and double
bonds (or cyclopropane rings) of the whole molecule [RCH(OH)CH(R)COOH], the P-unit
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K . K A N E D A A N D OTHERS
_
1
2
_
3
.
4
.
5
.. . . . . . . 6
7
8
9
10
.
1 1 1 2 1 3 14
Fig. 1 . AgNO, (10% v/v)-TLC of a-mycolic acid methyl esters from rapidly growing mycobacteria (a)
and slowly growing mycobacteria (b). Chromatograms were developed four times with n-hexanef
diethyl ether (94:6, v/v). (a) Lanes: 1, M . chitae CH-2; 2, M .fortuitum 181 12; 3, M . srnegmatis Takeo;
4, M . chelonae R-1; 5 , M . rhodesiae RHO-1; 6, M . diernhoferi 41002; 7 , M . phlei 14002; 8, M . parafortuiturn PA-1 ; 9, M .therrnoresistibile Lab.; 10, M . pulveris 33505; 1 1 , M.uaccae VA-1; 12, M . aururn
AU-1; 13, M . gilvum GI-1 ; 14, M . agri 90012. (b)Lanes: 1, 'M.novum'24020; 2, M . triviale 37014; 3, M .
terrae 38021 ;4, M . tuberculosis H 3 7 R ~5,;M .bovis Ravenel; 6, M . bovis BCG ;7, M.kansasiiLab.;8, M .
simiae 93001 ; 9, M . rnarinurn 08010; 10, M . gordonae T-12109; 1 1 , M.intracellulare 13542; 12, M . avium
11016; 13, M . ulcerans 28504.
[RCH(OH)] and the a-unit [CH(R')COOH] could be determined. For example, the mass
spectrum of one major GC peak of a-mycolic acids in M. uaccae (Fig. 3 ) showed [MI+ = 1194,
[M - 15]+= 1179 and [M - 90]+ = 1104, which corresponded to C,,-a-mycolic acids with two
double bonds (or cyclopropane rings); [A]+ = 841,813 and [B]+= 455,483, indicating that these
C,,-a-mycolic acids consisted of two different molecules, i.e., C54 :2(or sedia)(fl-~nit) C24 :o(aunit) and C52 :2(or 524ia)(P-unit) C24 ,o(a-unit). In every mycobacterial species, a-mycolic acids
possessed two double bonds (or the equivalent in cyclopropane rings). A double bond and a
cyclopropane ring in a-mycolic acids could not be differentiated from each other by the mass
spectrum only, because dicyclopropanoyl a-mycolic acid derivatives showed mass numbers
equivalent to the dienoic species.
GC or G C / M S anaIysis combined with the hydrogenation technique. To clarify whether the P-unit
of the a-mycolic acids possessed a cyclopropane ring or a double bond, we combined GC or
GC/MS analysis with the hydrogenation technique. When the a-mycolic acid derivatives of
M . chitae, which showed only a lower spot on AgN03-TLC (Fig. l), were analysed after
hydrogenation in neutral solvents the retention time of the G C peak became longer by about 0.7
carbon numbers and the mass spectral [M - 15]+ and [A]+ ions increased by four mass units,
showing that these a-mycolic acids were dienoic and that they received four hydrogens to
become saturated acids (Fig. 3). On the other hand, when a-mycolic acid derivatives of M.
vaccae, which showed only an upper spot on AgNO-TLC (Fig. l), were hydrogenated in the
neutral solvent, no change was observed. After hydrogenation in the acidic solvent, however,
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Mycobacterial mycolic acids
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Fig. 2. Gas chromatograms of TMS-ether derivatives of a’- and a-mycolic acid methyl esters from
rapidly growing mycobacteria, showing characteristic profiles. The carbon numbers of the esters in
each peak were determined from the mass spectra. (a),M . chitae CH-2; (b),M . smegmatis Rabinovitz;
(c), M . chelonae R-2; (d),M . fortuitum F-6; (e), M.fortuitum subsp. acetamidolyticurn E-11620; 0,M .
parafortuitum PA-4; (g), M . rhodesiae RHO-1 ; (h), M . uaccae VA-1; (i), M . aurum AU-1; 0,
M . agri
90002; (k), M . thermoresistibile Lab.
the retention time of the GC peak became shorter by about 1.5 carbon numbers and its mass
number of [M - 15]+ and [A]+ increased by four mass units with no change in the mass number
of [B]+, showing that the a-mycolic acids were dicyclopropanoyl and that they received four
hydrogens in the acidic solvent to cleave cyclopropane rings and to become dimethyl branched
saturated acids (Fig. 3). For M. phlei, which showed two spots, upper and lower, on AgN0,TLC (Fig. l), we examined the a-mycolic acids of each spot by mass spectrometry after
hydrogenation in the neutral solvent. Derivatives of the a-mycolic acids in the upper spot
showed no change, indicating that they were dicyclopropanoyl acids, but the mass spectral
[M - 15]+ and [A]+ ions from the lower spot increased by two mass units, indicating that they
were monocyclopropanoyl monoenoic acids (the lower spot also contained a small amount of
dienoic acids as indicated by coexistence of a slightly higher intensity mass spectral [A]+ ion
increased by four mass units) (Fig. 4). In M. diernhoferi, M. rhodesiae and M . parafortuitum, the
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Fig. 3. Gas chromatograms and mass spectra of the main GC peak with the corresponding carbon
skeleton structure of a-mycolic acid derivatives from M.chitae CH-2(a, b) and M.vaccae VA-1 (c-e)
before hydrogenation (a, c) after hydrogenation in chloroform/methanol(b,d ) or after hydrogenationin
glacial acetic acid (e).
upper spots on AgN03-TLC were also shown to be dicyclopropanoyl a-mycolates and the lower
spots to be composed of similar amounts of dienoic and monocyclopropanoyl monoenoic acid
derivatives from the two strong [M - 15]+ ions with increases of two and four mass units after
hydrogenation in the neutral solvent (data not shown). The upper spots in most slow growers
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Mycobacterial mycolic acids
400
740
750
830 840
2219
1190 1200 1210
mle
Fig. 4. Mass spectra of M. phlei 14002 C,,-a-mycolic acid derivatives from an upper (a, b) and a
lower (c, d ) spot obtained on AgN0,-TLC, before (a, c) and after (by d ) hydrogenation in
chloroform/methanol.
(Fig. 1) were shown to be dicyclopropanoyl acid derivatives and the lower spots in the M .
nonchromogenicum complex which includes ‘ M . novum’, M . terrae and M . triviale (Fig. 1) to be
dienoates. M . bovis BCG, exceptionally, contained a small amount of dienoic a-mycolates (lower
spot) (Fig. 1) in addition to the major dicyclopropanoyl species (upper spot).
Mass chromatography. Since the gas chromatographic peaks of mycobacterial a-mycolic acid
derivatives tended to overlap with their neighbours due to their relative broadness and the
relatively high contents of odd carbon-numbered acids (as described later), the molecular species
composition of a-mycolic acids was determined by measuring the area of each peak shown
independently on a mass chromatogram which monitored the mass number of [M - 15]+ions of
each carbon-number class of diunsaturated a-mycolic acids (Fig. 5). Data on the molecular
species composition of a-mycolic acids obtained by this method and the average carbon number
in representative mycobacterial species were reported previously (Kaneda et al., 1986b). There
was a marked specificity in the distribution of a-mycolic acid molecular species among the
mycobacteria. For example, the main peak of a-mycolic acids was C74 in M . vaccae, M .
parafortuium, etc., C76in M . fortuiturn, M . aurum, etc., C77in M . chitae, M . rwnchromogenicum,
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K. K A N E D A A N D O T H E R S
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Fig. 5 . Mass chromatograms of total (upper lower spots) a-mycolic acid derivatives from M.uaccae
VA-1 (a), M.chitue CH-2 (b) and M.phlei 14002 (c), the components of the upper (d) and lower (e) spots
on AgN03-TLC from the mycolates of the latter strain are also shown. The mass numbers shown on the
right correspond to the [M- I5]+ ions of TMS-ether derivatives of the dienoic or dicyclopropanoyl amycolic acid methyl esters whose carbon numbers are given in parentheses. The thick solid line is the
gas chromatogram. Thin solid lines and dotted lines are mass chromatograms of even and odd carbonnumbered acid derivatives, respectively. The carbon numbers C72-C80,shown at the bottom of the
figure indicate the relative retention of each unbranched dienoic or dicyclopropanoyl a-mycolic acid.
etc., C78in M. gilvum, M. bouis, etc., C79in M. terrae, M . triuiale, etc. and C,, in M. agri, M.
tuberculosis, etc. The molecular species composition of a given mycobacterial species did not
vary significantly as long as the growth conditions did not change. The proportion of even
carbon-numbered acids was characteristic to each species and was conveniently divided into
three groups. (1) Even acids up to 30%: M . terrae 38016* (25.2%); M . nonchromogenicum09003*
(26.0%); ‘M. novum’ 24018* (27.0%). (2) Even acids from 30 to 70%: M. smegmatis Jucho.*
(32.4%); M . chitae CH-2 (37.3%); M . chelonae 19009* (48.9%); M . rhodesiae RHO-1* (50-1%);
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Mycobacterial mycolic acids
222 1
M . phlei 14002 (57.8%); M . fortuitum 18001* (58.1%); M . parafortuitum PA-1 (61.1%); M .
diernhoferi 41002 (67-1%). (3) Even acids more than 70%: M. bovis BCG (72.7%); M . rnarinum
08010* (73.0%); M . pulveris 33505 (73.6%); M . vaccae VA-1 (76.4%); M . tuberculosis H 3 7 R ~ *
(77.4%); M . agri 90012* (81.3%); M . avium 11016* (82.0%); M . intracellulare 13023* (82.0%);
M . bovis Ravenel* (82.0%); M . thermoresistibile Lab.* (82.3%); M . aurum AU-1* (86.0%); M .
gordonae T-12109* (87.5%); M . gilvum GI-1 (89.0%); M . ulcerans 28504 (89.5%); and M.
kansasii Lab.* (94-3%). [The values for the species with an asterisk were calculated from the data
in our previous paper (Kaneda et al., 1986b); the remaining species were newly examined or reexamined in this study.]
Mass chromatograms, monitoring [M - 15]+ ions of a-mycolic acids of M. vaccae and M .
chitae, showed that the retention time of the odd carbon-numbered acids differed between these
two species; in M. vaccae, odd acids came essentially between the even acids, whereas in M .
chitae, odd acids appeared close to the retention time of an even acid derivative with one carbon
less (Fig. 5). Considering the data on the a-mycolic acids of M. smegmatis (Danielson & Gray,
1982), which shows a similar pattern to M. chitae, and the fact that the methyl-branched-chain
fatty acids appear just after the non-branched-chain parent fatty acids on the gas
chromatograms, we believe that the latter type of odd carbon-numbered a-mycolic acids
possesses one methyl branch on the straight alkyl portion (/?-unit).
We further examined the mass chromatograms of a-mycolic acid derivatives from an upper
spot (dicyclopropanoyl acids) and a lower spot (unsaturated acids) when there were two spots on
AgN03-TLC (Fig. I), as in M. phlei (Fig. 5 ) . There was a significant difference between these
two spots not only in the chemical structure but also in the molecular species composition. In M.
phlei, dicyclopropanoyl a-mycolic acids were composed mainly of even carbon-numbered acids
and the odd acids appeared between the even acids on the mass chromatograms, indicating that
there was no methyl branch on the /?-unit. The unsaturated acids (mainly monocyclopropanoyl
monoenoic acids as revealed by the hydrogenation experiment) were composed of similar
amounts of even and odd acids, and, from the similar retention time of the even acid derivatives
to those of the odd acid derivatives with one carbon less, the even acids possessed one methyl
branch on the /?-unit. The dicyclopropanoyl and unsaturated acids were distributed within a
similar carbon number range, and their average carbon numbers were also similar (Table 1).
The molecular species composition and the existence of a methyl branch on the P-unit was also
determined for other mycobacterial species which showed two spots for their mycolate
derivatives on AgN03-TLC (Table 1). Mycobacterial dicyclopropanoyl a-mycolic acids were
generally non-methyl-branched and consisted mainly of even acids; the proportion of even acids
was 76.4% in M. vaccae VA-1,76-5%in M . phlei and 82.2% in M. bovis BCG. On the other hand,
the unsaturated acids (dienoic acids and/or monocyclopropanoyl monoenoic acids) consisted
mainly of odd acids (the proportion of even acids was 21.1 % in M. parafortuitum PA-1,20-1% in
M . diernhoferi 41002, and less than 30% in the M. nonchromogenicum complex) or of a similar
amount of even and odd acids (the proportion of even acids was 46.7% in M . phlei 14002 and
37.3% in M. chitae CH-2), often possessing one methyl branch on the /?-unitof the even acids (M.
parafortuitum and M . phlei) or on that of odd acids (M. chitae, M. smegmatis, M .fortuitum and the
M . nonchromogenicum complex) (Table 1). In the case of mycobacterial species which possessed
a similar amount of both even acid-dominated dicyclopropanoyl acids and odd acid-dominated
unsaturated acids, as in M. phlei, M. parafortuitum, M . diernhoferi and M . rhodesiae, the
proportion of even carbon-numbered a-mycolic acids amounted to 40-60 %.
From the mass spectra of each molecular species (Fig. 3) or from mass chromatogram
monitoring of [B - 29]+ ions (426,454 and 482) the sizes of the chains in the a-position of the amycolic acids could be determined. Mycobacterial species were grouped according to whether
the a-unit was C22,Ct4 or C26.(1) Mycolic acids with essentially a Cz2-a-unit:M . parafortuitum
and M . diernhoferi. (2) Mycolic acids with C22-and C,,-a-units: M. phlei, M. rhodesiae, M .
gilvum, M . vaccae, M . aurum, M . pulveris and M . duvalii. (3) Mycolic acids with essentially a C24a-unit ; M. chelonae, M . smegmatis, M . chitae, M . fortuitum, ‘M.peregrinum’, M . porcinum, M .
thermoresistibile, M . agri, M . terrae, M . nonchrornogenicum, ‘M.novum’, M . triviale, M . avium, M .
intracellulare, M . kansasii, M . marinum, M . gordonae, M . ulcerans and M . shimoidei. (4) Mycolic
acids with a C2,-a-unit: M . simiae, M. bovis and M . tuberculosis.
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Species
M.bouis BCG
M . phlei 14002
M.parafo.rtuiturn PA-1
M.diernhoferi 41002
M .vaccae VA-1
M.chitae CH-3
Total
diene
(= lower)
Total
din
(= upper)
Total*
Upper
diA
Lower
monoA
diene
Total*
Upper
diA
Lower
monoA
diene
Total*
Upper
diA
Lower
monoA
Total*
Upper
diA
Lower
diene
Spot on
AgN0,-TLC
r
\
73
4.2
79
4.7
9-2 22.8
1.8 27.7
15-9 14-8
1-8 1-9 13-1
2.2 3.1 12.3
10-4
8.3 16.0 26-1 18.8 24.6
6.3 8-0 29.3 12.3 35.7
3.4 35.7 9.0 32.2 8.8
2.9
3-3
20.5
1 1.5
34.0
10.0
6.6
21.2
31.0
43-9
28-8
38.7
39.5
15.7
4.1 2.1
8-4
6.8
4.0
2.6
5.6
5.6
12.5
10.2
3.4
12.8
5.9
30-6
7.3 9.0 17-9 34.2 12.1 19.5
74
4.2 37-4 9-0 34.0
72
Carbon no. :
75 76 77 78
A
80
81
77.3
77-5
76.9
78.1
78-3
78.4
74.6
74-9
74-3
74.1
74.4
73-7
75-0
76.9
76-5
46-7
72.7
82-2
34.4
57.8
61.1
71.3
21.1
67.3
86-3
20-1
76-4
37.3
Percentage
proportion
of even
-,Average carboncarbon numbered
no.
acids
82
4.0
4.9
3-0
19.1 2.6
21.4 2-2 6.7
8-2 13.8
Molecular species composition (%)
5.9 18-1 11.9 25.0 11-1 21.1
2-3 18.4 3.8 35-6 5-0 29.1
14-3 6.4 34.0 7.2 26-0 6.5
71
+ lower spot.
-
-
-
-
+
r
t A methyl branch on even or odd carbon-numbered acids.
Total = upper
-
-
+ monuene +
-
-
-
-
-
-
Even Odd
A
Methyl
brancht
+ monuene +
+ monuene
L
Structure
Table 1. Structure and molecular species composition of the a-mycolic acid homologous series from each spot separated on AgN03-TLC
7c
[A
EP
4
U
0
>
>
z
n
U
z
>
7c
2223
Mycobacterial mycolic acids
Fig. 6. Mass spectra of the main peaks of a‘-mycolic acid derivatives from M . vaccae VA-1 (a, b) and M
forruitum 18001 (c, d ) before (a, c) and after (b, d ) hydrogenation in chloroform/methanol.
Analysis of a’mycolic acids
Several peaks of a’-mycolic acid derivatives with sizes ranging from CS8to C,o were clearly
observed on gas chromatograms (Fig. 2). In M. vaccae, the mass spectrum of the main peak
showed: [MI+ = 972, [M - 15]+ = 957 and [M - 90]+ = 882, which corresponded to C62-a’mycolic acids with one double bond (or a cyclopropane ring); [A]+ = 619, 591 and [B]+ = 455,
483, indicating that these C62-a’-mycolicacids contained C40:1(0r40-monon>(P-unit)
C2*,o(a-unit)
and C38:1(~~
3 ~ - m o n o n ~ ( ~ - ~C24:o(a-unit)
nit)
(Fig. 6). In M. fortuitum the main component was
determined as c 6 8 : 2(or 68-d,,)-a’-mycolicacids with a C24 ,o-a-unit. After hydrogenation in the
neutral solvent, the a’-mycolic acids both of M . vaccae and of M .fortuitum showed mass numbers
equivalent to those of the saturated acids, indicating that these two a’-mycolic acids were
monoenoic and dienoic, respectively (Fig. 6). In this study, a’-mycolic acids of every
mycobacterial species were shown to have one double bond except in M.fortuitum and related
taxa ( M .fortuiturn subsp. acetamidolyticum, ‘M.peregrinum’ and M . porcinum), which possessed
two double bonds. Although the C66,68a’-mycolic acids of M . smegrnatis, as reported by Etkmadi
et al. (1967), and the C6,-a‘-mycolic acids of M . chelonae consisted of a significant amount of
dienoic acids as revealed by mass chromatography monitoring the [M - 15]+ ions of both
dienoic and monoenoic a’-mycolic acids (data not shown), these dienoic acids only made up a
small proportion of the total a’-mycolic acids. Furthermore, these monoenoic or dienoic a’mycolic acids did not have a methyl branch on their P-unit (deduced from the fact that the odd
carbon-numbered acids were always situated between even acids on the mass chromatograms
produced by monitoring [M - 15]+ ions). From the mass chromatograms of a’-mycolic acids
monitoring [B - 29]+ ions of C22-, C24- and C,,-a-unit-containing mycolic acids, the
proportions of these three homologous series of a’-mycolic acids were shown to be similar to
those of the a-mycolic acids from the same bacterial species.
The molecular species composition of a’-mycolic acids was determined by measuring each
peak area on gas chromatograms or mass chromatograms, and the average carbon number was
calculated (Table 2). The average carbon number of a’-mycolic acids varied from species to
species; it was about 60 in M . parafortuitum and M . vaccae, about 62 in M . chitae, M . duvalii, M .
aurum and M. pulueris, about 64 in M. smegmatis, M . chelonae, M . shimoidei and M .
thermoresistibile, about 66 in M. simiae and about 68 in M. fortuitum, M . fortuitum subsp.
acetamidolyticum, ‘ M .peregrinum’ and M . agri. Mass chromatographic analysis revealed that a’mycolic acids from every mycobacterial species were composed mainly of even carbon-
+
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+
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1.0
M . parafortuitum PA-1
M . vaccae VA-1
M . chitae CH-2
M . duvalii DU-2
M . pulveris 33505
M . aurum AU-1
M . smegmatis Rabino.
M . chelonae R-1
M . thermoresistibile Lab.
M . shimoidei 43501
M . simiae 93001
M . fortuitum 18001
' M . peregrimun' PE- 1
M . agri 90002
57
23.4
29-5
1-3
3-4
58
60
61
3-7
59-8
49.3
23.2
16-1
1-1 15-1 4.7
10.8
4.3
59
65
1.0
1.4
56.1
18.4
42.0
31-1
36.8 7-2 27.3 3-2
43.8
34.7
39-5
41-8
11.1
83.9
15-2
44-2
10-4 5.0 43-2 8.0
18.5
3-4
1.7
3-0 4.4
14.6
19.8
Carbon no.:
62
63
64
A
67
68
69
1.0
6.3
1.1
4-6
9.6
1.1
11.7
2.7
5.0
32-8
4.1
25-7 2.5 5-1
72.5
9.0
25.2
63-5
29.9
63.8
21.0 13-6 50-6 5.3
66
Molecular species composition (%)*
a-Unit
22
22, 24
24 (22)t
22, 24
22, 24
22, 24
24
24
24
24
26 (24)t
24
7.9
24
4.6
24
2-1
70
\
34-42
36-44
3H2
36-44
3742
38-44
36-44
3842
36-44
3 8 4
38-44
40-46
40-46
4246
59-7
59-9
61-9
62.5
62-5
62-9
63.4
63.9
64-4
64.6
65-8
67.5
67-4
67.3
Average
carbon
no.
B-Unit
t A small but a significant proportion of mycolic acids with the structure shown in parentheses coexist.
* Values shown in bold are the main components of a given species.
56
Species
f
1
1
1
1
1
1
1 (2)t
1
1
1
1
2
2
1
Double
bond
no.
With the exception of M . pulveris, M . shimoidei and M . agri, species containing less than 15% of odd carbon-numbered a'-mycolic acids are not listed.
Table 2 . Molecular species composition of a'-mycolic acids in rnycobacteria
P
N
N
N
Mycobacterial mycolic acids
2225
numbered acids. The proportion of odd carbon-numbered a’-mycolic acids was generally up to
about 20% except in M. agri: M. vaccae VA-1 and M .fortuitum 18001 (trace amount); M. simiae
93001 (6.5%); M . chelonae 22011 (7.8%); M . chitae CH-3 (8.2%); M . duvalii 29506 (9.7%); M .
parafortuitum PA-1 (9.8 %); M. smegmatis Rabinovitz (1 1-3%); M . thermoresistibile Lab.
(1 1.3%); M. shimoidei 43501 (15-5%); M. pulveris 33505 (16-2%) and M. agri 90002 (23-3%).
Variation in the structure and the molecular species composition of a-mycolic acids from 16
rapid growers and 14 slow growers allows mycobacterial species to be divided into five groups,
A-E, as indicated in Table 3.
DISCUSSION
Mycobacterial a-mycolic acids possess two dicyclopropanoyl rings, one cyclopropane ring
plus one double bond or two double bonds on the Q-unit (Minnikin, 1982; Daffk et al., 1983).
These three different homologous series of a-mycolic acids were identified from the mass ion
shift after hydrogenation in neutral or acidic solvents. The upper spot on AgN03-TLC was
shown to be dicyclopropane and the lower one was olefinic acid derivatives - monocyclopropanoyl monoenoic acids and/or dienoic acids. It is uncertain why the latter two homologous series of
a-mycolic acids could not be clearly separated on AgN0,-TLC. However, Minnikin (1982)
reported that one of the two double bonds on the odd carbon-numbered, mono-methyl branched
(or monocyclopropanoyl) a-mycolic acids were not cis monoenoic but trans monoenoic. Since it
is well known that trans monoenoic fatty acids migrate between the saturated and the cis
monounsaturated acids on AgN03-TLC, a-mycolic acids may also behave similarly. In this
study, by examining the a-mycolic acids of each upper and lower spot on AgN0,-TLC, it was
clearly shown that a significant difference existed in the molecular species composition between
the dicyclopropanoyl acids and the olefinic acids. Dicyclopropanoyl a-mycolic acids were
composed mainly of even carbon-numbered acids and possessed no methyl branch on the P-unit,
whereas olefinic acids were composed either of predominantly odd carbon-numbered acids or of
a similar amount of odd and even carbon-numbered acids, often possessing one methyl branch
on the P-unit. The result obtained here (that a methyl branch often existed in the a-mycolic acids,
possessing one or two double bonds) is in accordance with the structural formula of a-mycolic
acids presented by Minnikin et al. (1984). This is reasonable because a methyl branch is
considered to be introduced on the adjacent carbon of a double bond during its cis to trans
conversion (Minnikin, 1982).
Many rapid growers produce a’-mycolic acids which have shorter main chain Q-units than
those in the a-mycolic acids. In the slow growers, only two species (M. shimoidei and M . simiae)
contained this type of mycolic acid subclass. However, there was no difference in the structure or
the composition of the a’-mycolic acids from rapid growers and those from slow growers. The a’mycolic acids of M. fortuitum and related taxa were characteristically dienoic, whereas in many
other species they were monoenoic. These a’-mycolic acids, both monoenoic and dienoic,
possessed no methyl branch on the /?-unit and contained only a small amount of odd carbonnumbered acids (M. agri, M. shimoidei and M . pulveris possessed relatively more odd carbonnumbered acids, but always less than 30% of the total a’-mycolic acids). The carbon chain length
of a’-mycolic acids in groups B and C was generally shorter (up to c6z) than in group A (more
than C64).
It has been shown that a’-mycolic acids are not merely small forms of a-mycolic acids, but
have rather independent structures, e.g. (1) in groups B and C, the a’-mycolic acids were
monoenoic, whereas the a-mycolic acids were mainly dicyclopropanoyl derivatives ; (2) in group
A, the odd carbon-numbered a-mycolic acids were methyl-branched, but the a’-mycolic acids
were not; (3) the proportions of odd carbon-numbered acids were very low in a’-mycolic acids;
and (4) there were no obvious correlations in the carbon chain length between a- and a’-mycolic
acids within the same bacterial species - M. chitae, M . smegmatis and M . thermoresistibile,which
contained longer a-mycolic acids (c,
possessed shorter a’-mycoliC acids (c62-64) and,
conversely, M. fortuitum and ‘M. peregrinum’, which possessed intermediate sized a-mycolic
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Rapid growers
Group A
M. chelonae
M . smegmatis
M . chitae
M . fortuitum
‘ M . peregrinum’
M . porcinurn
M . fortuitum subsp.
acetamidoly ticum
Group B
M . diernhoferi
M . phlei
M . rhodesiae
M . parafortuitum
Group C-1
M . gilvum
M. vaccae
M . aurum
M . pulveris
M . duvalii
Group C-2
M.thermoresistibile
M . agri
Species
r
B- Unit *
Structure
I
I
I, 11, I11
I, 11, 11‘
I, 11, 11’, 111, 111’
I, 11, 11’, 111, 111’
111, 111‘
111, 111’
111, 111’
111, 111‘
111, 111’
111, 111’
111, 111‘
(main component)
I
(WIl
24
24
80
80
78
74
76
78
76
22,
22,
22,
22,
22,
24
24
24
24
24
74
78
74
74
24
24
24
24
77
77
77
76
76
77
78
3
0
0
0
0
0
0
ND
X
X
X
X
Proportion of
even acid$
Composition
Main
peak
22
22, 24
22, 24
22
24
24
24
a-Unit?
\
A
a-Mycolic acids
-
22
-
IV
IV
IV
IV
IV
IV
IV
24
24
22,
22,
22,
22,
24
24
24
24
-
-
-
-
-
24
24
24 (22)Il
24
24
24
24
IV
IV
IV
I11
I11
I11
I11
Composition
>
64
68
60
62
62
62
-
60
-
62
68
68
68
68
64
64
Main
peak
Proportion of
even acid$
&
L
a’-Mycolic acid
a-Unitt
Structure
B-U nit*
f
Table 3 . Chemical structure and molecular species composition of a- and a‘-mycolic acids in mycobacteria
N
S
P
S
S
N
Chromogenesisi
m
N
N
N
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I
I
I
ND
I
I
I
I
I
I
111,
111,
111,
111,
111'
111'
111'
111'
26 (24)l)
26
26
82
78
80
0
0
0
-
-
IV
-
-
26 (24111
-
-
66
I I
CH ( CH2 ),CH-CH
I I
CH2)xCH3
II '
. CH3 ( CH2)]
CH-CH(
CHC2,)H=
CH,
OH COOH
CHCH( CH2 ),CH-CH(CH2)xCH3
0
-
Q N, Nonchromogenic; s, scotochromogenic; P, photochromogenic.
11 A small but a significant proportion of mycolic acids with the structure shown in parentheses coexist.
So, >70%; x, 3&70%; XX, <30%.
( CH2 )xCH3
OH COOH
CH2),CH-CH(
OH COOH
CH2),CH=CH(
OH COOH
Not determined.
The structural formulae are cited from Minnikin et al. (1984) as shown below.
ND;
Carbon number.
I V. CH3 ( CH2 )lCH=
I I . CH3(CH2 )]CH-CH(
Slow growers
Group D
M . terrae
M . nonchromogenicurn
' M . navum'
M . triviale
Group E-1
M . avium
M . intracellulare
M . kansasii
M.gordonae
M . marinum
M . ulcerans
M . shimoidei
Group E-2
M . simiue
M . bovis
M . tuberculosis
N
N
N
N
2228
K . K A N E D A A N D OTHERS
acids (C,,), contained longer or’-mycolic acids (C68).
The proportions of the C22-and C2,-a-units
were, however, similar between the or- and or’-mycolic acids from the same bacterial species. The
compositions of the or-units in the mycolic acids analysed here (Table 3) are in good agreement
with the results obtained by pyrolysis gas chromatography (Kusaka & Mori, 1986).
Chromogenicity can be used to divide rapid growers into two groups. The chromogenic
species include many saprophytic mycobacteria isolated from soil or water and the nonchromogens may include several opportunistic pathogens such as M . fortuitum and M . chelonae
besides other non-pathogenic species (Goodfellow & Wayne, 1982). The grouping of rapid
growers based on mycolic acid patterns was concordant with chromogenicity. Groups B and C
included many chromogenic species but the mycobacteria in group A were all nonchromogenic.
There is, however, no obvious correlation between the biosynthesis of carotenoid pigments and
mycolic acids. Furthermore, Groups B and C included several nonchromogenic species, e.g. M .
diernhojeri, M . pufveris and M . ugri, and there was no correlation between chromogenicity and
mycolic acid patterns in the slow growers. Although chromogenicity is often used as a
convenient criterion for identifying or classifying mycobacterial species, the significance of
pigment production in the physiology of mycobacterial species and its taxonomic value are not
yet clear (Tsukamura, 1963 ; Minnikin, 1982). Furthermore, during repeated culture,
nonchromogenic species often acquire a slight chromogenicity which sometimes causes
confusion in identification. The grouping of mycobacteria by mycolic acid patterns is more
reliable because mycolic acids are always major structural components of acid-fast bacteria and
many characteristic enzymes must be involved in their biosynthesis (Minnikin, 1982;
Takayama & Qureshi, 1984; Lacave et af., 1987). Furthermore mycolic acid patterns are
relatively constant if the cultural conditions do not vary.
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